Highly pathogenic avian H5N1 influenza viruses circulating endemically in poultry of southeast Asia have recently been disseminated across the globe by wild migratory birds. These avian influenzas are also able to infect humans, and during the past decade have caused several outbreaks of severe illness with high mortality. There is concern that H5N1 bird flu viruses may in the future evolve to forms that are easily transmitted to, and among, people. There is also fear that vaccines directed against past and currently circulating H5 influenzas may not be effective against future strains of the virus due to inevitable antigenic drift and shift. The overall goal of this project is to develop hemagglutinin antigens that elicit cross protective, rather than type-specific, humoral antibody responses against a broad spectrum of evolving H5N1 influenza strains. Our work will be based on the hypothesis that ablating actively evolving, well-characterized primary antigenic determinants on the H5 hemagglutinin will redirect immune responses to regions of the molecule that are conserved and normally not antigenic. These conserved "secondary" epitopes are more likely to be preserved in future epidemic (and possibly pandemic) strains, than are the actively evolving, antigenic drift epitopes targeted by conventional vaccines. Therefore, the recombinant hemagglutinins we develop may be useful as "universal" immunogens and provide broad H5 protection in vaccination and immunotherapy protocols. Specific Aim 1 is to design, express and purify milligram amounts of several hemagglutinins. Design of these x-HAs will be based on a wealth of mapping information from natural drift and escape mutants of H5 and related influenzas, and the crystal structure of the Viet/1203/04 H5 HA. To neutralize the primary antigenic determinants, primary antigenic determinant residues identified by escape mutants will be replaced with amino acids that are present at low frequency in the Discotope database of antigen-antibody interfaces. Expression / purification strategies will follow established protocols for producing properly folded hemagglutinin molecules. Primary antigenic determinant site knockout will be verified by screening with a panel of mapped mAbs to the A/Viet/1203/04 HA. Specific Aim 2 is to prepare mouse antisera against control and x-HA hemagglutinins and assay their virus neutralization capacity using an influenza hemagglutinin pseudotyped lentiviral vector reporter assay. We predict that x-HA.s on which all primary antigenic determinants have been knocked out will elicit antibodies to evolutionarily conserved "secondary" antigenic determinants, and will be able to neutralize reporter viruses representing different H5 influenza clades. Antisera containing neutralizing antibodies will be useful for development of passive immunotherapeutics against H5 influenzas, and the x-HA antigens used to generate them will be candidates for the development of universal vaccines to stimulate the production of broadly protective humoral immunity to H5 influenzas. PUBLIC HEALTH RELEVANCE: Because conventional influenza vaccines stimulate immune responses against parts of the virus that evolve steadily over time, it is necessary to produce new seasonal flu vaccines each year. The goal of this work is to develop avian influenza vaccines that are not aimed at the parts of the viral hemagglutinin that change, and that instead target hemagglutinin structures which remain constant over time. The antigen molecules developed in this work may be useful for protecting people from pandemic strains of avian influenza, and the strategy used to develop them may also be applicable to generating broadly protective "universal" vaccines against seasonal flus and other pathogens that evade the immune system by changing their surface structures.